The ability of ligands to induce biphasic (or multiphasic) protein denaturation should be independent of the means used to effect protein denaturation. Thus, we began a study of the effect of ligands on the guanidine HC1-induced unfolding of human albumin. We anticipated that biphasic denaturation should occur in the presence of subsaturating levels of a high affinity ligand. A preliminary experiment with undefatted human albumin, which contained approximately 1.4 mol of bound high-affinity, endogenous, long-chain fatty acid/mol of albumin monomer and which corresponds to approximately 15% saturation, showed biphasic denaturation when monitored by UV protein difference spectroscopy and by changes in intrinsic protein fluorescence. However, on defatting human albumin and subjecting this protein to denaturation with guanidine HC1 in the absence of ligand, we found that this ligand-free protein also undergoes biphasic denaturation; this behavior had been demonstrated earlier for bovine albumin by Khan et al. using urea [J. Biochem. 102, 313-317 (1987)]. Clearly this biphasic denaturation is not ligand-induced but rather relates to the domain substructure of the protein. We propose to look for a smaller protein that in the absence of ligand unfolds as a single cooperative unit when denaturing with strong denaturant but that has a ligand for which it has sufficient affinity to show ligand-induced biphasic denaturation when unfolding with strong denaturant.